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Welcome to the website of ConStruct, the consortium for structural biology in Heidelberg.

Mission

Structural biology has revolutionized our understanding of cellular architecture, and the function and mechanism of its components. ConStruct enhances interactions and exchange between Heidelberg groups with strong interests in structural biology. ConStruct HD bundles these activities to pursue integrated structural biology approaches to explore e.g. structures and dynamics of molecular machines and motors, supramolecular complexes, structural networks, and in unraveling their mode of action.

ConStruct is tightly linked with CellNetworks. ConStruct combines core expertise in crystallography, electron microscopy, NMR spectroscopy, computation, and structural biochemistry. It reaches out to promote interactions with other members of CellNetworks.

Organisation

ConStruct is shaped by its funding core members (J. Briggs, EMBL; B. Bukau, ZMBH; T. Carlomagno, EMBL; A. Meinhart, MPI; R. Russell, Bioquant; K. Scheffzek, EMBL; I. Schlichting, MPI; R. Schröder, CellNetworks / Bioquant; I. Sinning, BZH), who meet every two months after the ConStruct seminar. The initiative is headed and managed by I. Schlichting and R. Schröder. ConStruct is open to all interested parties, in particular the members of CellNetworks.

Members

We are interested in the mechanisms of assembly and budding of enveloped viruses and coated vesicles. We aim to understand how proteins collect together the cargo of the virus or vesicle, and define and manipulate the shape of the membrane to cause budding. To explore these questions we are studying a range of different cellular and viral specimens using cryo-electron microscopy and tomography. A particular emphasis of our research is the structure and life-cycle of asymmetric membrane viruses such as HIV.

Methods

Cryo-electron microscopy and tomography techniques are particularly appropriate for studying vesicles and viruses because they allow membrane topology to be observed in the native state, while maintaining information about the structure and arrangement of associated proteins. Computational image processing and three-dimensional reconstructions are used to extract and interpret this information. We also have and interest in the development and optimization of novel microscopy and image processing methods, including those for correlative fluorescence and electron microscopy.

The laboratory focuses on studying 1) structure-activity and dynamics-activity relationships of RNP complexes and catalytic RNAs involved in RNA processing; 2) the interaction of small drugs with cellular receptors. In the first area our work aims at describing the features of RNA-protein recognition in RNP complex enzymes and at characterizing the structural basis for their function. In the second area of research, we develop both computational and experimental tools to access the structure of large receptors in complex with small molecules that regulate their function.

Methods

For the structural study in solution we use nuclear magnetic resonance (NMR) spectroscopy in combination with a wide range of biochemical and biophysical techniques (small angle scattering, X-ray crystallography, calorimetry, etc.). Recent advances in the NMR methodology and instrumentation have allowed overcoming traditional size limitations and have made NMR a very powerful technique, in particular for the investigation of highly dynamic, partially inhomogeneous molecules and complexes.

Cytochrome P450s (P450s) are a superfamily of oxidative haemoproteins that are widespread in nature and can catalyse a vast array of oxidative transformations. We study a group of P450s that are selective for their substrates bound to carrier proteins: small domains found in large biosynthetic machineries such as non ribosomal peptide synthetases. These P450s play crucial roles in the biosyntheses of many important natural products including the glycopeptide antibiotics and have the potential to act as biocatalysts in future chemical syntheses.

Methods

We work in the field of chemical biology. Our group uses organic chemistry to synthesise small molecules and peptides that we use to modify proteins of interest, either through direct chemical or enzyme-catalysed means. We then apply protein crystallography, various chromatographic methods and biochemical/biophysical techniques to study structural and mechanistic aspects of these modified proteins and their interactions. Additionally, we utilise various expression systems to obtain the proteins that we require to perform our studies.

Hsp70 and Hsp90 chaperones cooperatively assist maturation and control stability and activity of a large number of client proteins, many of which are signaling kinases and transcription factors. We are interested in the molecular mechanism of the Hsp70 and Hsp90 chaperones, how they are regulated themselves by cochaperones, and how they control their client proteins. In particular, we investigate the conformational dynamics of these proteins to understand the conformation-function relationships.

Methods

We use hydrogen-exchange mass spectrometry to determine the secondary structure flexibility and fluorescence spectroscopy including fluorescence energy resonance transfer to monitor domain movements. In addition, we use a large variety of biochemical and biophysical techniques including circular dichroism spectroscopy, FT-IR spectroscopy, isothermal titration calorimetry, FACS and fluorescence microscopy to analyze wild type and mutant proteins in vitro and in vivo.

RNA is heavily modified and quality checks are performed throughout the life cycle of an RNA molecule. We study macromolecular machines that regulate RNA maturation at the 3’ end. In particular, we are interested in the mechanism of complex assembly and regulatory events triggered by phosphorylation and RNA recognition. The second topic is the mechanism of inhibition and activation of bacterial Toxin-Antitoxin assemblies that provoke suicide of individual bacteria thereby supporting the entire remaining cellular population during stress condition and infection.

Methods

We use advanced crystallography techniques in order to obtain three dimensional structures of these macromolecular assemblies and machines. Furthermore, we apply various biophysical and biochemical techniques such as Fluorescence Anisotropy, Isothermal Titration Calorimetry, UV and CD spectroscopy etc. to investigate functional mechanisms such as complex assembly, enzymatic activity. In case of Toxin-Antitoxin assemblies, we use different microbiology techniques to combine ex vivo and in vivo results.

We are studying molecular mechanisms of transcriptional regulation in eukaryotes, where DNA is packaged into chromatin. We are interested how sequence-specific transcription factors, co-activators and general transcription factors interact to recruit RNA polymerase to the transcription start site in the context of chromatin. We are also analyzing the molecular architecture of chromatin modifying and remodeling complexes and how they interact with chromatin templates. Finally, we try to understand how chromatin structure and dynamics affects transcription initiation and elongation processes.

Methods

A particular focus of the laboratory is the structural and biochemical analysis of multi-protein complexes involved in eukaryotic transcription and chromatin modification. We use X-ray crystallography as principal techniques combined with single-particle electron microscopy. In addition, we use complementary biophysical approaches (isothermal calorimetry, CD, static and dynamic light scattering, analytical ultracentrifugation) and various biochemical and cellular biology approaches.

Technical Platform

The Müller group is the EMBL coordinator of P-CUBE (Infrastructure for Protein Production Platforms). The program offers general access to European users to the most advanced techniques in cloning, expression, protein characterisation and crystallisation. Further information can be found under: www.pcube-eu.

We study blue light photoreceptors containing flavin chromophores to understand how the energy of a photon absorbed by the sensor domain is transformed into a structural change that (in)activates an associated effector domain. We study light-regulated phosphodiesterases, cyclases, and transcription factors with the ultimate goal to design sensors that can be used for cell biological applications (optogenetics). FELs provide ultra-short and -intense coherent X-ray pulses that allow watching the making and breaking of molecular bonds and are predicted to "outrun" radiation damage, thereby allowing to study nanocrystals and macromolecular assemblies. We are part of an international team exploring biological applications.

Methods

We use standard molecular biology, biochemistry, enzymology , and a wide range of biophysical methods to characterize the photoreceptor proteins. For structural characterization, we use and develop X-ray scattering and diffraction techniques (conventional crystallography, static and time-resolved small and wide angle X-ray scattering, femtosecond nano-crystallallography, coherent imaging) using home, synchrotron and Free Electron Laser sources.

Membrane proteins comprise more than 25% of the cellular proteome and their function depends on insertion into the correct target membrane. Membrane proteins utilize predominantly the universally conserved co-translational delivery pathway of the signal recognition particle (SRP), while a subset of membrane proteins utilize the GET pathway (guided entry of tail-anchored proteins). We are interested in the molecular mechanisms of the SRP and Get pathways, and their regulation by NTP-binding proteins. We study membrane insertases as well as chaperones and enzymes that act on nascent chains to unravel the early steps in the life of a membrane protein.

Methods

We use advanced X-ray crystallography techniques as our key method – together with biochemical and biophysical methods (e.g. ITC, UV and CD spectroscopy, amide hydrogen deuterium exchange with mass spectrometry, EPR spectroscopy, cryo-electron microscopy …). The combination of in vivo and in vitro assays allows to unravel the molecular mechanisms and regulation of our target machines. State of the art expression and purification techniques are the basis of our studies. We have established a HTP-crystallization platform that is open to external users.